人胎肺间充质干细胞分离培养条件及向心肌细胞诱导分化的可能关联  

Isolation,culture and differentiation of human fetal lung mesenchymal stem cells into cardiomyocytes

作　　者：贾文文[1] 华进联[1] 于海生[1] 杨玉艾[1] 赵婷[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学国家干细胞工程技术研究中心陕西分中心,陕西省杨凌市712100

出　　处：《中国组织工程研究与临床康复》2007年第3期439-442,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：教育部重大专项(03160)~~

摘　　要：目的:观察分离培养得到人胎肺间充质干细胞的条件,并分析诱导其转分化为心肌细胞的可能性及其相关因素。方法:实验于2003-01/2005-12在西北农林科技大学陕西省干细胞工程技术研究中心完成。取10周龄人工流产胎儿肺组织,0.25%胰蛋白酶消化,经培养得到人胎肺间充质干细胞。观察细胞形态及增殖,测细胞生长曲线。将人胎肺间充质干细胞以1.8×104/孔接种于明胶预处理的24孔板中。加Dulbecco修正Eagle’s基础培养液培养24h后弃去培养液,磷酸盐缓冲液(-)洗两三次。每14孔为1组,分别加入Dulbecco修正Eagle’s基础培养液和先进的Dulbecco修正Eagle’s(分别添加2%、5%、8%和10%新生牛血清)5种不同的培养液,其后方法同上,每天选任意两孔计数,求平均值。用流式细胞仪对所分离第8代细胞进行以下细胞表面标志检测:CD45、CD11a、CD14、CD90、CD34、CD71、CD25、CD105、CD117、CD166和CD44。人胎肺间充质干细胞向心肌细胞诱导分化:将第12代人胎肺间充质干细胞以500个/孔接种于明胶预处理的24孔板中。每6孔为1组,分别在Dulbecco修正Eagle’s基础培养液中添加0、5、10、20μmol/L5-氮胞苷,每组中每2孔为一诱导时间段,分别诱导培养24,48,72h后换成Dulbecco修正Eagle’s基础培养液,以后每3d换液1次。观察细胞在各组中变化情况,诱导12d后固定进行糖元染色和α平滑肌肌动蛋白免疫组化染色。结果:成功分离了人胎肺间充质干细胞,细胞已扩增至26代,流式细胞仪检测CD25、CD105、CD166和CD44表达阳性;CD71(39.1%)和CD117(29.8%)弱阳性。Dulbecco修正Eagle’s培养液比先进的Dulbecco修正Eagle’s培养液更适合于这类细胞生长。经10μmol/L5-氮胞苷诱导48h后,糖元染色、心肌α平滑肌肌动蛋白免疫组化染色阳性细胞率达60%以上。结论:①人胎肺间充质干细胞在Dulbecco修正Eagle’s基础培养液中能够大量扩增。②并表达CD25,CD105,CD166�AIM： To observe the isolation and culture of human fetal lung mesenchymal stem cells （MSCs）, and analyze the potential and related factors-to the differentiation into cardiomyocytes. METHODS; Experiment was completed in Shaanxi Center of Stem Cell Engineering and Technology, Northwest Science and Technology University of Agriculture and Forestry from January 2003 to December 2005. Lung of 10 weeks old fetus was dissected and digested by 0.25% Trypsin. The proliferation and morphology of cells were determined, then the growth curve of cells were calculated. Human fetal lung MSCs were incubated into 24-pore plate that was pretreated with gelatin, each containing 1.8×10^4 cells. Dulbecco＇s Modified Eagle＇s Medium （DMEM） was used to culture cells for 24 hours and then removed, followed by washed 2-3 times with phosphate buffer. Afterwards the cells ware allocated into 5 groups and culture in DMEM and advanced DMEM （ADMEM）, which was supplemented with 2%, 5%, 8% and 10% new-born calf serum respectively. The mean value of cells was studied by cell counting. Flow cytometry was used to determine the surface markers of passage-8 MSCs, including CD45, CD11a, CD14, CD90, CD34, CD71, CD25, CD105, CD117, CD166 and CD44. Cells at passage 12 were inoculated into 24-pore plate that was pretreated with gelatin, with 500 cells in each pore. The cells were grouped as 6 pores in each and then cultured in DMEM medium with 0, 5, 10 and 20 p.mol/L 5-azacytidine for another 24, 48 and 72 hours, and every 2 pores in one group were taken as one period of cardiomyocyte inducing culture. The medium was removed every 3 days. The effects of different serum concentrations and medium to cultured cells were observed. The differentiated cells were identified by the Periodic acid Schiff staining （PAS） and α-actin immunohistochemistry staining. RESULTS： Human fetal lung MSCs were obtained successfully and subcultured to the 26^th passage. It was showed that the cells were positive by FITC labeled CD25, CD105, CD166, CD44,

关 键 词：胚胎 间质干细胞 心肌 细胞学 

分 类 号：R394.2[医药卫生—医学遗传学]